It is now widely recognized that astrocytes (astroglia) play a central role in controlling excitability of neurons and contribute to the cellular mechanisms associated with learning and the formation of new memories. The plasticity of learning and memory formation involve changes in dendritic spines at glutamatergic synapses on neurons, and astrocytes form tripartite synaptic structures at a majority of mature spines. Astrocytes regulate extracellular glutamate levels and secrete gliotransmitters, neurotrophic factors, and proteases that affect the function and structure of the tripartite synaptic structure. It is this close physical localization of astroglia with glutamatergic synapses that places the astroglial process in a position to control neuronal excitability and synaptic plasticity. Moreover, the presence of astrocytes at glutamatergic synapses modulated the structure of the spine and controls NMDA receptor-dependent metaplasticity of hippocampal CA1 pyramidal neurons. It is well- established that chronic exposure of alcohol to hippocampal neurons increases the number of NMDA receptors at glutamatergic synapses, and it has also been suggested that this increase may promote actin-dependent increases in the size of the spine. These changes are suggested to contribute to alcohol dependence and tolerance. However, it is unknown what role astrocytes play in regulating the structural and functional plasticity of glutamatergic synapses associated with chronic alcohol exposure and withdrawal. In this exploratory R21 application, we will examine chronic alcohol-induced structural remodeling of the tripartite synapse. The over-arching hypothesis is that alcohol exposure induces the release of certain proteases from both neurons and astroglia that control spine-astroglial structural and functional plasticity. Three specific aims are proposed that will test our proposed model of alcohol- induced plasticity of tripartite synaptic structure: Aim 1 will test the hypothesis that acute and chronic in-vitro alcohol exposure alters the structural dynamics of the tripartite synapse in a live-cell preparation;Aim 2 will test the hypothesis that chronic in-vivo ethanol exposure alters the morphology and density of tripartite synapses;and Aim 3 will test the hypothesis that ethanol-induced structural plasticity of the tripartite synapse is associated with the release of proteases that act upon the synaptic extracellular matrix. These studies will utilize novel diolistic dye loading procedures and lentiviral infection of fluorescent proteins to visualize tripartite synaptic structures coupled with confocal imaging and sophisticated 3D-image analysis of alcohol-induced alterations in spine-astrocyte structures. The broad, long term objectives of this proposal are to determine if astrocytes contribute to alcohol-induced plasticity of glutamatergic synapses and whether astrocytes can be identified as a novel therapeutic target for the treatment of alcohol abuse and dependence. PUBLIC HEALTH RELEVANCE: Alcoholism is characterized by craving for alcohol and compulsive alcohol-seeking behavior. Thus, determining the processes by which alcohol exposure leads to aberrant and inappropriate synaptic connections of the brain may lead to novel approaches to effective treatments of alcoholism and alcohol related disorders.